Background: The importance of genetic exchange between species (i.e. introgression of genes from one species into another through hybrid intermediates) is a constant subject of debate among evolutionary biologists. While some claim genetic exchange is pervasive, others claim that it is extremely rare. Currently, there is little data quantitatively supporting either of these scenarios. Broad, long-term objective: The proposed study of naturally occurring hybrid zones will quantify the prevalence of genetic exchange between species to understand the frequency and evolutionary consequences of gene exchange. The proposal takes advantage of these natural experiments in hybrid zones to find admixed genomes with which I can map the genetics of complex behavior traits that differ between the hybridizing species. Since genetic mapping of behavior traits has proven very difficult by conventional means, admixture mapping provides a unique opportunity to make progress on these important questions. Specific aims: Aim 1 of the study is to map the prevalence of introgression in six natural hybrid zones, four of which are documented here for the first time. Aim 2 will used admixed flies from a hybrid zone to map genes underlying the ecologically important trait of temperature preference. Aim 3 will use admixed flies from another hybrid zone to map genes underlying the complex behavioral trait of mate preference. Method: Fruit flies from the genus Drosophila can be collected in their natural habitat and also maintained under laboratory conditions, and have an unmatched arsenal of molecular genetic and genomic tools. This proposal presents a novel model for identifying introgressed alleles in the genome, an approach that is only feasible in hybridizing organisms for which both parental species have high quality genome assemblies. The genes identified in these admixture analyses will be functionally validated with CRISPR-mediated gene replacements. Preliminary results for admixture mapping and genome editing are both promising. Health-relatedness: Admixture mapping in humans is a primary approach used for the identification of disease-causing gene variants. However, follow up experiments to test the function of genes correlated with disease cannot be performed in humans. Admixture mapping protocols have therefore never been validated end-to-end from the population level at which variants correlated with disease are identified, to the functional molecular genetic level where the phenotypic state is manifested. This proposal is innovative because it develops new computational methods to identify genomic regions with complex ancestry, and then connects these genomic patterns to measurable phenotypic differences via genome editing. Impact: The results from this study will provide the first broad view of patterns of introgression in cases where species interbreed in nature, and will leverage those patterns of admixture to dissect the genetic basis of behavioral differences between species that cause reproductive isolation.